The Pyrogram shows the mutation (nucleotide 34, indicated with an arrow) with the "Sequence to Analyze" GNTGRCGTAGGYA targeting base 2 in codon 12 (nucleotide 35). The yellow color indicates that this sequence is unexpected and needs to be checked.

Reanalysis of the data in figure "GGT to AGT mutation in base 1 of codon 12".

The mutation GGT → AGT was reanalyzed with the "Sequence to Analyze" NGTGRCGTAGGYA targeting base 1 in codon 12 (nucleotide 34). With this new targeted sequence to analyze, the software rates the assay as one of good quality, confirming the quantification of the mutation.

Principle of Pyrosequencing — step 1.

Principle of Pyrosequencing — step 2.

Principle of Pyrosequencing — step 3.

Principle of Pyrosequencing — step 4.

Principle of Pyrosequencing — step 5.

Workflow solutions.

The components of the PyroMark Q24 MDx System are designed to make the Pyrosequencing workflow straightforward and efficient. Each step, from assay design to PCR amplification and preparation of sequencing templates, is supported by software, kits, reagents, and sample prep instrumentation optimized for Pyrosequencing.

Fully integrated system.

Though small in size, the PyroMark Q24 MDx manages all steps necessary to rapidly analyze up to 24 samples. Simply load samples and reagents, upload the run file, and walk away. The PyroMark Q24 MDx dispenses reagents and nucleotides to each well with precision. Light signals emitted are detected by 24 CCD sensors — one sensor per well — thereby eliminating signal crossover.

Easy data management.

The PyroMark Q24 MDx is designed as a stand-alone instrument, which makes it easy to place anywhere in the lab. Data are stored on the instrument hard drive and can be viewed on the instrument screen during a run. Additionally, all files are stored on the supplied USB stick, giving the user the flexibility to analyze data on any computer with PyroMark Q24 MDx Software installed.

Efficient template prep.

The PyroMark Q24 MDx Vacuum Workstation enables conversion of PCR products into the single-stranded DNA needed as template for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process is carried out for 24 samples in parallel and takes only a few minutes.

The right instrument for your needs.

The PyroMark Q24 MDx combines ease of use, analysis versatility, and superior detection sensitivity to meet the needs of your diagnostic work. For research purposes, we also offer the PyroMark Q96 ID and PyroMark Q96 MD that can process up to 96 samples in a single run. The PyroMark Q96 MD can additionally be automated to run ten 96-well plates.

Performance

Pyrosequencing technology enables accurate and sensitive quantification of genetic and epigenetic DNA variations by providing highly reliable sequence data. It allows the identification of novel mutations, as well as detection of aberrant DNA methylation patterns present at low levels.

The therascreen KRAS Pyro Kit is a good example of the informative analysis provided by the therascreen Pyro Kits and the PyroMark Q24 MDx instrument. The KRAS gene is mutated in approximately 35% of metastatic colorectal cancer (CRC) patients. Studies have shown that KRAS mutation testing can better define which CRC patients will benefit from treatment with epidermal growth factor receptor (EGFR) inhibiting monoclonal antibodies, such as panitumumab and cetuximab.

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 MDx is a fully integrated system that provides real-time sequence information and is highly suited for genetic and epigenetic analysis. The system includes the PyroMark Q24 MDx Instrument, PyroMark Q24 MDx Vacuum Workstation, PyroMark Q24 MDx Software 2.0, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo. Sample preparation solutions are also available to enable preparation of single-stranded DNA using the PyroMark Q24 MDx Vacuum Workstation.

Steps of the Pyrosequencing reaction:

Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure "Principle of Pyrosequencing — step 1").

Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure "Principle of Pyrosequencing — step 2").

Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure "Principle of Pyrosequencing — step 3").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure "Principle of Pyrosequencing — step 5").

Dedicated IVD-validated assays

QIAGEN offers an expanding suite of IVD-validated assays to be used with the PyroMark Q24 MDx. Currently, these kits include important cancer-related mutations:

Product

For quantitative measurement of mutations in

therascreen KRAS Pyro Kit

codons 12, 13, and 61 of the human KRAS gene

therascreen BRAF Pyro Kit

codons 600 and 464-469 of the human BRAF gene

therascreen EGFR Pyro Kit

codons 719, 768, 790, 858, and 861 and exon 19 of the human EGFR gene

therascreen NRAS Pyro Kit

codons 12, 13, and 61 of the human NRAS gene

Procedure

From PCR product to single-stranded template ready for sequencing — up to 24 samples can be prepared in parallel using the PyroMark Q24 MDx Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling, and the actual hands-on time is less than 5 minutes.

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Gold reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

The PyroMark Q24 MDx is suitable for in vitro diagnostic applications in Europe.

Genetic analysis comprises multiple applications to analyze differences in genomic DNA, including mutation detection and SNP typing. PyroMark Q24 MDx facilitates accurate and highly sensitive mutational analysis of any gene of interest and enables quantification of allele representation in mixed cell populations. The system can be used for self-validated assays for oncology studies and for analysis of epigenetic markers in methylation studies, or it can be used with dedicated IVD-validated assays available from QIAGEN

Software

PyroMark Q24 MDx Software, installed on a PC, enables comprehensive analysis of your results. The software contains two analysis modes: CpG and AQ (allele quantification). Both modes can be used to analyze samples on the same plate, enabling different types of samples to be run at the same time. The AQ mode can be used for analyzing single and multivariable positions, as well as di-, tri- , and tetra- allelic mutations. The CpG mode enables analysis of multiple consecutive CpG sites and provides a built-in control for the bisulfite treatment.

This version is compatible with Windows 7 and Windows 10 (64 bit) operating systems. This software may only be downloaded by registered users with a valid PyroMark Q24 MDx software license and registered PyroMark Q24 MDx instrument. If you do not have a valid software license, contact your QIAGEN sales representative.